Free Radical Biology & Medicine, Vol. 37, No. 4, pp. 511–520, 2004Copyright D 2004 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2004.04.025

Original Contribution

ASCORBATE DISTRIBUTION DURING HIBERNATION IS INDEPENDENT OF

ASCORBATE REDOX STATE

YI LONG MA,* MARGARET E. RICE,y MEI LAN CHAO,y PATRICIA M. RIVERA,* HUIWEN W. ZHAO,* AUSTIN P. ROSS,*

XIONGWEI ZHU,z MARK A. SMITH,z and KELLY L. DREW *,z

*Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK, USA; yDepartment of Physiology and Neuroscience,New York University School of Medicine, New York, NY, USA; and z Institute of Pathology,

Case Western Reserve University, Cleveland, OH, USA

(Received 19 November 2003; Revised 16 April 2004; Accepted 22 April 2004)

Available online 25 May 2004

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Abstract—Distribution of ascorbate into tissues is an essential process in ascorbate antioxidant defense. Hibernating

animals are studied as a model of tolerance to ischemia–reperfusion because of their tolerance to fluctuations in blood

flow associated with prolonged torpor and periodic arousal episodes. Throughout hibernation, plasma ascorbate

concentration ([Asc]p) repetitively increases during torpor, then falls during periodic arousal bouts. We previously

proposed that high [Asc]p provides a ready source of antioxidant protection for distribution to the central nervous system

and peripheral tissues during arousal. Here we tested whether deliberate oxidation of plasma ascorbate by intravenous

administration of ascorbate oxidase (AO), prior to arousal, compromised tissue levels of ascorbate or the other water-

soluble antioxidants, glutathione (GSH) and urate. Although AO decreased [Asc]p to below the level of detection during

torpor and after arousal, ascorbate oxidation did not decrease post-arousal tissue levels of reduced ascorbate, glutathione,

or urate in any tissue examined, except liver. The data imply that ascorbate is taken up equally well into brain and other

tissues as either ascorbate or its oxidized product dehydroascorbate, with subsequent intracellular reduction of

dehydroascorbate. Lack of effect of ascorbate oxidation on tissue levels of GSH or urate indicates that dehydroascorbate

uptake and reduction do not compromise tissue concentrations of these other water-soluble antioxidants. Thus, we show

equal availability of reduced and oxidized plasma ascorbate during metabolically demanding thermogenesis and

reperfusion associated with arousal from hibernation. D 2004 Elsevier Inc. All rights reserved.

Keywords—Vitamin C, Ascorbic acid, Reperfusion, Hypothermia, Torpor, Dehydroascorbate, Glutathione, Urate, Uric

acid, Free radicals

INTRODUCTION

Ascorbate is the most important antioxidant in plasma

[1] and has been shown to minimize ischemic damage

in models of cerebral ischemia–reperfusion, both in

vivo [2–5] and in vitro [6,7]. Hibernation is neuro-

protective [8] and is studied as a model of tolerance to

ischemia, reperfusion, neurodegenerative disease, and

ress correspondence to: Dr. K. L. Drew, Institute of Arctic

y, Alaska Basic Neuroscience Program, PO Box 757000,

sity of Alaska Fairbanks, Fairbanks, AK 99775-7000, USA;

07) 474-6050; E-mail: ffkld@uaf.edu.

511

brain injury [9,10]. During hibernation, ascorbate con-

centration increases 4-fold in plasma ([Asc]p) and

doubles in cerebralspinal fluid [11]. On arousal, [Asc]pdecreases to levels seen in euthermic animals (37jCbody temperature), at the same time as O2 consumption

[12] and cerebral blood flow [13] reach maximum

levels. We have proposed previously that during the

300-fold surge in oxidative metabolism that is neces-

sary to rewarm from 2 to 37jC, ascorbate is redis-

tributed to metabolically active tissues, including the

brain, where it provides protection against oxidative

stress [12]. To address this hypothesis, we assessed

whether oxidation of ascorbate in plasma prior to

resurgence of oxidative metabolism during arousal from

Y. L. Ma et al.512

hibernation compromises tissue ascorbate content. Our

results indicate that oxidizing ascorbate in the plasma

compartment prior to arousal from hibernation does not

affect tissue ascorbate content after arousal in brain or

any tissue studied (except liver), nor does it affect

tissue concentrations of the other water-soluble anti-

oxidants, glutathione (GSH) and urate, in brain or any

tissue examined.

MATERIALS AND METHODS

Animal handling

All procedures were approved by the Institutional

Animal Care and Use Committee of the University of

Alaska Fairbanks and were in accordance with National

Research Council’s Guide for the Care and Use of

Laboratory Animals. Arctic ground squirrels (AGS,

Spermophilus parryii) were trapped during mid-July in

the northern foothills of the Brooks Range, AK, USA,

approximately 30 miles south of the Toolik Field Station

of the University of Alaska, Fairbanks (68j 38V W,

elevation 809 m), and transported to Fairbanks. The

squirrels were housed individually at 18jC, under a

natural light cycle for 64jC latitude, and fed rodent

chow, sunflower seeds, and fresh carrots and apples ad

libidum until mid-September. They were then moved to

an environmental chamber set to an ambient temperature

(Ta) of 2jC and 4:20 h light:dark cycle. Animal experi-

ments were conducted December through April. Hiber-

nation was assessed from core body temperature or by

inactivity (indicated by the lack of disturbance of wood

shavings placed on an animal’s back 24 h previously).

Deep hibernation was verified by rectal temperature at

the time of euthanasia.

Surgical procedures

Telemetry transmitters, used to monitor core body

temperature, and indwelling femoral arterial and venous

cannulas were implanted in all AGS treated with AO or

saline. Prior to surgery, AGS were placed at 21jC and

left overnight to arouse from hibernation. General

anesthesia induced with halothane (Halocarbon Lab,

Riveredge, NJ, USA) at 5% was maintained at 2.5–

3% mixed with 100% medical grade O2 at a flow rate

of 1.5 l/min. Surgery was performed under strictly

aseptic conditions. Body temperature was kept between

36.5 and 37.5jC with a fluid-filled heating pad (Omni

Medical Equipment, Cincinnati, OH, USA) throughout

the surgery. Cannulas were inserted into the femoral

artery and vein (Tygon tubing; 0.375 mm i.d., 0.75 mm

o.d.; Norton, Akron, OH, USA), passed subcutaneously

and dorsally to an incision at the back of the neck, and

secured with suture as described previously [12]. Pre-

calibrated, wax-coated telemetry transmitters (Model

VM-FH, 2 cm disk, Mini Mitter Co., Inc., Sunriver,

OR, USA) were implanted intraperitoneally via a

midline incision through linea alba, and sealed with

three layers of sutures. Animals were allowed 1 day

postoperative recovery at 18jC before being returned

to the environmental chamber to resume hibernation.

Ascorbate oxidase experiments

To determine the effect of ascorbate redox state

during arousal on postarousal tissue levels of reduced

ascorbate, GSH, and urate, plasma ascorbate was

deliberately oxidized with AO administered by intra-

venous injection. AO (EC 1.10.3.3), isolated from

zucchini, oxidizes ascorbate to dehydroascorbate

[14,15]. Control AGS received an equal i.v. volume

of saline (0.9% NaCl) vehicle. For these experiments,

AO (96 IU/kg in a volume of 0.6 ml/kg saline) or

vehicle was administered immediately before arousal

from hibernation during four consecutive bouts of

torpor with mean F SEM torpor bout lengths of

6.1 F 0.88 days (AO group) and 5.5 F 0.54 days

(saline group). In each of the four episodes, arterial

blood was sampled immediately prior to i.v. injection

(T0) and 30 min after injection (T1). An additional

blood sample was collected after the fourth arousal

bout (4T2) when an animal had reached a body tem-

perature of at least 35jC (see Fig. 1). At each sampling

point, approximately 150 Al of blood was collected into

heparinized capillary tubes and spun for 5 min in a

microcapillary centrifuge, and three 20 Al samples of

plasma were collected and immediately frozen on dry

ice. These samples were stored at �80jC until ascor-

bate analysis.

Tissue collection

Tissues were collected immediately after arterial

blood was sampled on the fourth arousal bout. Tissue

was sampled from four groups of animals as described

above (euthermic, hibernating, aroused-AO, and

aroused-saline). Before initial sampling in euthermic

and aroused AGS, animals were lightly anesthetized

with halothane (5% halothane mixed with O2 delivered

at 1.5 l/min); hibernators were not initially anesthetized.

Rectal temperatures were measured with a copper–

constantan thermocouple. After decapitation, brains

and peripheral tissues (spleen, liver, kidney, heart, skel-

etal muscle, brown fat, white fat, adrenal, small intes-

tine) were removed immediately, dissected, and frozen

in liquid N2. Brain samples were taken from frontal

cortex, hippocampus, and cerebellum, and frozen in

liquid N2 as in previous studies [11,12]. Frozen tissue

samples were stored at �80jC. Time from decapitation

to freezing was less than 10 min.

Fig. 1. Core body temperature over time in an individual AGS illustrates time of blood sampling and AO or saline injection. Arterialblood was sampled at T0 followed immediately by intravenous injection of AO (96 IU/kg) or saline. Thirty minutes later (T1), blood wassampled again, before body temperature began to increase. Emergence from hibernation was initiated during blood sampling and wasrepeated four times to mimic natural periodic arousal from hibernation. Numbers 1 through 4 indicate arousal bouts. Blood was sampledafter the fourth injection, and body temperature reached 35jC (4T2) just prior to tissue collection. Torpor bout length averaged (FSEM)5.7 F 0.4 days and did not differ between AO- and saline-treated groups.

Ascorbate distribution and redox state 513

HPLC analysis

Tissue samples. On the day of analysis, tissues

(typically 10 mg) were weighed and sonicated while

still frozen in ice-cold, deoxygenated eluent (see be-

low). Samples were then centrifuged, and the superna-

tant was injected directly onto the HPLC system.

HPLC analysis was based on methods described pre-

viously [12,16] using a gold amalgam electrode for

ascorbate and GSH and a glassy carbon electrode for

urate. Eluent contained 16 mM monochloroacetic acid,

with 150 mg L�1 EDTA and 300 mg L�1 myristyl

dimethylbenzylammonium chloride, pH 5, and up to

20% methanol. Retention time for urate is similar to

that for GSH under these conditions, however, GSH is

not detected on the carbon electrode and thus does not

interfere with urate analysis. Standards were prepared

in ice-cold, deoxygenated eluent at regular intervals

throughout the analysis. Tissue contents of ascorbate,

urate, and GSH are expressed as micromoles per gram

wet weight of tissue.

Plasma samples. On the day of analysis, plasma sam-

ples were diluted 1:4 in ice cold, deoxygenated eluent (see

above) and spun at 14,000 rpm for 1 min, and 10 Al of thesupernatant was again injected directly onto the HPLC.

Reduced plasma [Asc]p and urate ([Urate]p) were ana-

lyzed simultaneously using methods similar to those for

tissue samples with an ESA 5100A coulometric detector

with the analytical cell (Model 5011; ESA, Inc., Chelm-

ford, MA, USA) set at 0.4 V versus Ag/AgCl. Plasma

levels of GSH were not determined. The limit of detection

for ascorbate (signal-to noise ratio of 3:1) with this assay

was 1.0 AM per injection (i.e., 4 AM [Asc]p).

Chemicals

All chemicals were analytical grade. Myristyl dime-

thylbenzylammonium chloride (Myris-100) was a gift

from Jame Fine Chemicals, (Bound Brook, NJ). EDTA,

monochloroacetic acid (MCA), ascorbate, uric acid, and

GSH were purchased from Sigma–Aldrich (St. Louis,

MO, USA). Ascorbate oxidase (L-ascorbate oxidoreduc-

tase, EC 1.10.3.3) was purchased from Calzyme Labo-

ratories, Inc (San Luis Obispo, CA, USA). All glassware

used for HPLC analysis was treated with concentrated

HNO3, followed by thorough rinsing with deionized

water [17].

Statistics

All plasma and tissue samples were analyzed by

HPLC in triplicate. Subsequent statistical analyses were

performed on means of triplicates with n equal to the

number of animals. Plasma urate concentrations were

compared using repeated-measures ANOVA, paired t

test, or t test where indicated. Tissue contents of ascor-

Y. L. Ma et al.514

bate, urate, and glutathione in peripheral tissues were

compared using one-way ANOVA and Tukey post-hoc

comparisons. Differences in ascorbate content among

brain regions were analyzed using two-way ANOVA

followed by pairwise multiple comparisons (Tukey test)

(SigmaStat, SPSS Science, Chicago, IL, USA). Data are

expressed as group means F SEM. The criterion for

statistical significance was p < .05.

RESULTS

Effect of AO on [Asc]p

The effect of AO treatment on [Asc]p and [Urate]p is

shown in Fig. 2. By 30 min after AO injection (T1),

[Asc]p was below the detection limit in 14 of 16 samples

(Fig. 2A) (1T1 through 4T1 for each of four animals).

Although five animals received AO through patent

venous cannulas, intermittent plasma sampling was not

possible in one animal because the arterial cannula was

temporarily blocked). [Asc]p was also below the limit of

detection in all AO-treated animals sampled at 4T2immediately prior to tissue collection. Thus, enzymatic

Fig. 2. Effects of intravenous AO injection on [Asc]p and [Urate]p. (A)Thirty minutes after AO injection (T1), plasma ascorbate declined tobelow-detectable levels in the majority of samples. Although [Asc]pincreased again prior to the next injection, low [Asc]p at 4T2 indicatesthat [Asc]p remained low during arousal from hibernation. Decrease in[Asc]p in the saline group at 4T2 illustrates the typical decline in [Asc]pseen during arousal from hibernation. (B) The difference in [Urate]pbetween AO- and saline-treated groups at 4T2 was not statisticallysignificant ( p = .110, t test, n = 4–5).

oxidation of [Asc]p persisted after arousal (4T2 sampling

point), whereas in saline-treated control AGS, [Asc]preturned to a level (mean F SEM: 47.3F14.8 AM, n =

5) typical of euthermic animals [11,12].

We have suggested previously that [Urate]p may be an

indicator of oxidative stress [12]. Notably, AO adminis-

tration did not cause an increase in [Urate]p (Fig. 2B). On

the other hand, there was a tendency for [Urate]p to

increase after arousal from hibernation (at 4T2) in control

AGS, but not in AO-treated animals. The difference in

[Urate]p between groups at this sampling point was not

significant ( p = .110, t test, n = 4–5). Furthermore, no

significant difference was observed in [Urate]p analyzed

using a repeated-measures ANOVA or a paired t test to

compare [Urate]p sampled at 4T1 (prior to arousal) and

4T2 (after arousal) within saline-treated animals ( p =

.229, paired t test). Although some animals showed a

pronounced increase in [Urate]p, concentration decreased

in two of five animals after arousal. The tendency for

[Urate]p to increase at 4T2 in saline-treated AGS contrasts

with previous results in which [Urate]p peaked at the

same time as oxygen consumption and returned to basal

levels before core Tb reached 35jC (Fig. 2) [12]. The

large degree of variability in [Urate]p at 4T2 (Fig. 2B)

suggests the increase reflects variability in the time

course of the transient rise in [Urate]p rather than a

specific affect of AO.

Effect of hibernation, arousal, and [Asc]p oxidation on

brain ascorbate, GSH, and urate

Depletion of [Asc]p by AO had no effect on brain

ascorbate, GSH, or UA content in any brain region exam-

ined (Fig. 3). Regarding effects of hibernation and arousal,

ascorbate levels in brain were greatest in animals after

arousal, whether treated with saline or AO (Fig. 3A) (main

effect of group, p < .001). Brain ascorbate levels were

similar in aroused-saline and aroused-AO groups, but

differed from both euthermic and hibernation groups,

(Tukey test, p < .05 euthermic = hibernating < aroused-

saline = aroused-AO, n = 5). Although statistical compar-

ison indicates an overall increase in brain ascorbate levels,

results (Fig. 3A) suggest the increase in brain ascorbate was

restricted to frontal cortex. Nonetheless, the interaction

between treatment and brain region was not significant.

Similar analysis of brain GSH levels showed no

effect of hibernation state or arousal with or without

AO, although there were some regional differences in

absolute level, with higher GSH levels in hippocampus

than in frontal cortex or cerebellum (Fig. 3B) (main

effect of region, p < .001, n = 5; Tukey test comparing

regions, p < .05). Paradoxically, hibernation was asso-

ciated with a pronounced increase in urate levels, with

significantly higher levels during hibernation than in any

other state (Fig. 3C) (main effects of group, region, and

Fig. 3. AO treatment had no effect on (A) ascorbate, (B) GSH, or (C) urate levels in cerebellum (CB), frontal cortex (FC), orhippocampus (HPC). Regardless of treatment, ascorbate levels were greatest in brain following arousal (main effect of group, p < .001followed by Tukey test, p < .05 euthermic = hibernating < arousal-saline = arousal-AO, n = 5; interaction term was not significant sostatistical comparisons reflect combination of data across all brain regions and can not be represented by horizontal lines). Urate levelswere significantly increased during hibernation (main effect of group, region, and group � region, p < .001, n = 5; Tukey test comparinggroups, p < .05). Horizontal lines indicate significant differences between groups within each brain region; data shown are means FSEM (n = 5). Core body temperatures at time of tissue collection were (mean F SEM) 35.5 F 0.7, 2.8 F 0.2, 35.6 F 0.3, and 35.7 F0.4jC for euthermic, hibernating, arousal-saline (saline), and arousal-AO (AO) groups of animals.

Ascorbate distribution and redox state 515

group x region, p < .001, n = 5; Tukey test comparing

groups, p < .05).

Effect of hibernation, arousal, and [Asc]p oxidation on

ascorbate, GSH, and urate levels in peripheral tissues

Oxidation of plasma ascorbate by AO did not affect

the levels of reduced ascorbate in any tissue except

liver. In liver, where ascorbate is synthesized in these

animals, ascorbate levels decreased during hibernation,

then exceeded euthermic levels after arousal (Fig. 4A),

consistent with previous studies [12]. In liver, AO

attenuated the rebound in liver ascorbate content seen

after arousal in saline-treated controls (main effect of

group, p < .01, n = 5; Tukey test, p < .05 for all

Fig. 4. AO affected tissue ascorbate levels (A) only in liver where, in the saline treated group, ascorbate declined in hibernation, thensurpassed euthermic levels after arousal. AO treatment significantly attenuated the rebound in liver ascorbate after arousal (main effect ofgroup, p < .01, n = 5; Tukey test, p < .05 for all pairwise comparisons except AO vs. euthermic). AO increased GSH content in brownfat ( p < .05, AO vs saline, n = 5). Connected horizontal lines indicate other significant differences ( p < .05) between groups (means FSEM, n = 5) and reflect the decrease in tissue urate concentrations during hibernation. See Fig. 3 legend for core body temperatures attime of tissue collection.

Y. L. Ma et al.516

pairwise comparisons except AO vs euthermic). Oxi-

dation of plasma ascorbate by AO did not decrease

GSH or urate content in any tissue, although it did

produce a small, but significant increase in GSH

content in brown fat ( p < .05, AO vs saline) (Figs.

4B and 4C). Peripheral levels of urate fell during

Ascorbate distribution and redox state 517

hibernation, in contrast to the significant increase in

urate that occurred in brain (Fig. 4C).

DISCUSSION

Maintenance of tissue ascorbate levels by reduced or

oxidized plasma ascorbate

Ascorbate, an essential component of the antioxidant

network, is synthesized in the liver in mammals and

transported in plasma to all other tissues, where it is then

concentrated in the intracellular compartment by active

uptake [18]. Ascorbate is the most important antioxidant

in plasma [1] and has been shown to minimize ischemic

damage in models of cerebral ischemia–reperfusion,

both in vivo [3–5] and in vitro [6]. During hibernation,

ascorbate concentration increases 4-fold in plasma

([Asc]p) and doubles in cerebralspinal fluid [11]. On

arousal, [Asc]p declines to levels seen in euthermic

animals (37jC body temperature), and the rate of fall

peaks at the time of maximal O2 consumption [12] and

cerebral blood flow [13]. We have proposed that ascor-

bate is redistributed to vulnerable tissues during arousal

where it serves as an antioxidant and cytoprotectant. In

the present study, we asked if oxidizing ascorbate in the

plasma compartment prior to arousal from hibernation

would affect tissue ascorbate concentrations. We show

that recovery of tissue ascorbate content during arousal

does not require plasma ascorbate to remain in the

reduced form ([Asc]p). After arousal, tissue levels of

[Asc]p were the same in AO- and saline-treated AGS in

all tissues examined except liver. This suggests that

ascorbate and its oxidation product, dehydroascorbate

(DHA), are taken up equally well into tissues, with ready

reduction of DHA back to ascorbate by GSH and other

thiols [19,20] and by GSH-dependent dehydroascorbate

reductase [21,22]. The finding that the post-arousal tissue

levels of reduced GSH and urate were the same in AO-

and saline-treated animals argues that intracellular anti-

oxidant capacity is sufficient to allow for reduction of

DHA without compromising concentrations of these

other water-soluble antioxidants.

During arousal in AGS, ascorbate is presumably taken

up by active transport. In peripheral tissues, this uptake is

mediated by SVCT1 or SVCT2 depending on the organ

examined [23]. In the central nervous system, uptake of

reduced ascorbate occurs exclusively via SVCT2, first

from plasma via the choroid plexus (the site of CSF

production), then from the extracellular fluid into neu-

rons [18,23]; glial cells do not appear to express either

SVCT1 or SVCT2 [23,24]. Additionally, DHA can be

taken up in most cells via facilitated diffusion of DHA

through glucose transporters GLUT 1, GLUT 3, and

GLUT 4 [25–27]. Other studies have shown that if

sodium-dependent uptake of ascorbate becomes limiting,

such as after ischemia, uptake of DHA via glucose

transporters may enhance ascorbate distribution and

cytoprotection [5]. The results reported here suggest that

during arousal from hibernation, ascorbate and DHA are

equally well distributed to provide cytoprotection in

brain and other tissues studied.

In liver, ascorbate content returned to euthermic levels

after arousal in AO-treated animals, but did not rebound

to exceed euthermic levels as it did in saline-treated

controls. Although liver is the site of ascorbate synthesis

in mammals [28], hepatic levels are maintained by active

uptake, as indicated by the high density of SVCT1 in

liver [23] and the ready uptake of ascorbate into hep-

atocytes [29] and liver microsomes [30]. Results suggest

that in liver, uptake of ascorbate during arousal exceeds

that of DHA, most likely because of a high density of

SVCT1. A direct effect of AO on ascorbate synthesis in

liver is unlikely, as AO is a 70.4 kDa protein that would

be trapped in the plasma compartment after intravenous

injection.

AO treatment did not compromise tissue content of

ascorbate, GSH, or urate in other peripheral tissues,

such as spleen and brown fat, where ascorbate de-

creased during hibernation but returned to euthermic

levels on arousal. These results suggest, that as in brain,

DHA in peripheral tissues is taken up and subsequently

reduced to ascorbate without affecting concentrations of

other water-soluble antioxidants.

Redox balance during hibernation and after arousal

Tissue levels of ascorbate and GSH reflect oxidative

status of the tissue such that decreases in either one or

more of these antioxidants would be consistent with

decreased antioxidant capacity and oxidative stress.

Ascorbate in brain is tightly regulated [18] and fluctuates

little throughout hibernation and arousal [12]. A trend for

brain tissue levels of ascorbate to increase after arousal

observed previously [12] was statistically significant in

this independent set of samples. This increase could be

derived, in part, from ascorbate stores in cerebral spinal

fluid that double during hibernation [11]. Although it is

unclear if the slight increase in brain ascorbate provides a

physiologically relevant increase in antioxidant capacity,

preservation of brain ascorbate levels is consistent with a

lack of oxidative stress during hibernation or arousal.

Similarly, maintenance of GSH levels in brain is consis-

tent with redox homeostasis; however, because levels of

GSSG (and thus total GSH equivalents) were not mea-

sured in the present study, the question of GSH redox

balance remains open and requires further study.

Curiously, brain urate levels increased dramatically

during hibernation, consistent with previous results [12].

Reactive oxygen species rapidly convert xanthine dehy-

Y. L. Ma et al.518

drogenase to xanthine oxidase, the enzyme responsible

for metabolism of xanthine to urate. For this reason

transient increases in urate concentrations have been

interpreted as evidence of increased oxidative stress

[12]. Elevations in brain urate concentrations during

hibernation may likewise reflect increased oxidative

stress. Alternatively, increases in urate concentration in

brain may reflect enhanced nucleotide metabolism or

decreased urate clearance from brain tissue. More direct

and comprehensive measures of oxidative stress in brain

across the hibernation cycle are necessary to confirm

redox status during hibernation and arousal.

GSH decreased in spleen, liver, and brown fat during

hibernation, consistent with a shift toward a more oxi-

dized environment. Although preliminary studies [31,32]

have not revealed other evidence of oxidative stress in

these tissues during hibernation, these tissues warrant

further study. Carey et al. reported that the GSH/GSSG

ratio decreases 5-fold in intestinal mucosa during hiber-

nation in 13-lined ground squirrels (Spermophilus tride-

cemlineatus), an effect attributed to a 50% decrease in

GSSG reductase activity and subsequent increase in

GSSG [33].

Role of ascorbate as an antioxidant during arousal from

hibernation

Ascorbate is an essential water-soluble antioxidant

that has been shown to be an effective scavenger of

reactive oxygen species [18]. Although controversy over

a neuroprotective role of ascorbate stems from evidence

of pro-oxidant properties in vitro [34], the in vivo

findings reported here provide evidence of ascorbate

redistribution during arousal from hibernation and sug-

gest this antioxidant may protect tissues from oxidative

stress during arousal.

Frei et al. previously reported that in plasma, neither

protein thiols, a-tocopherol, nor urate can prevent initia-

tion of lipid peroxidation and formation of micromolar

concentrations of lipid hydroperoxides induced by

aqueous peroxyl radicals in human plasma [1]. Rather,

these antioxidants merely lower the rate at which lipid

peroxidation occurs. In contrast, ascorbate was found to

be the most effective aqueous-phase antioxidant under

experimental conditions of oxidative stress. Ascorbate not

only completely protected plasma lipids from peroxida-

tive damage, but also spared a-tocopherol, urate, and

bilirubin [1]. These data led Frei et al. to conclude that

ascorbate is an outstanding antioxidant in human plasma.

Ascorbate has selective antioxidant properties that

make it unique among the antioxidant network. For

example, ascorbate limits peroxynitrite formation by

reacting selectively with superoxide, which spares NO

signaling, and helps maintain enhanced vascular integrity

[35,36]. Interestingly, Haung et al. have suggested that

DHA may also provide vascular protection [5]. In the

brain, ascorbate selectively scavenges hydroxyl radicals

formed from hydrogen peroxide (H2O2) to prevent neu-

rotoxicity [37], while sparing H2O2 signaling pathways

[38]. Definitive assessment of neuroprotective effects of

ascorbate during arousal from hibernation, as well as the

relative roles of ascorbate and DHA, requires specific

inhibitors, which are not yet available. SVCT2 knockout

mice die at birth, in part from intraparenchymal brain

hemorrhage that cannot be accounted for by a scorbutic

effect [36].

Evidence of redistribution of ascorbate during arousal,

regardless of ascorbate redox state, supports our model

that enhanced antioxidant defense during hibernation is

an important cytoprotective component of hibernation

physiology. Hibernating animals tolerate extreme fluctu-

ation in brain and body temperature, cerebral blood flow,

and other physiological parameters associated with het-

erothermy [9]. Moreover, these animals endure a variety

of events including traumatic brain injury [8], hypoxia

[39], and oxygen-glucose deprivation [40] better than

homeothermic mammals such as rats and humans. Un-

derstanding neuroprotective aspects of hibernation phy-

siology thus has potential to translate into improved

therapies and strategies for treating cerebral ischemia,

brain injury, and neurological disease. Results reported

here demonstrate that ascorbate is distributed to tissues

equally well in either the reduced or oxidized state

without compromising concentrations of other water

soluble antioxidants. Thus, from a human therapeutic

perspective, where ascorbate must be provided exoge-

nously, administration of ascorbate or DHA should be

equally efficacious and distinguished only by patholo-

gical conditions that could influence distribution and

uptake. Moreover, our findings emphasize that depletion

of [Asc]p alone, without consideration of tissue levels of

ascorbate is a poor indicator of ascorbate availability. In

addition to reports for protection from reperfusion injury

by ascorbate, and DHA discussed above, there is ample

evidence for neuroprotection by other antioxidants in

other forms of central nervous system injury [41–43], as

well as in neurodegenerative diseases [44–46]. In the

human population, low plasma antioxidant activity is

associated with greater neurological impairment after

central nervous system insult [47] and a lifelong dietary

pattern poor in antioxidants correlates with a higher

propensity for neurological disease [48]. Moreover, low

dietary intake of ascorbate and other antioxidants is a risk

factor for morbidity and mortality from heart disease

[49], whereas ascorbate supplementation leads to im-

provement in endothelial function in a variety of cardiac

pathologies [50].

We show here that elevated plasma ascorbate levels

during hibernation provide an important source of anti-

Ascorbate distribution and redox state 519

oxidant in hibernating mammals. These findings should

guide use of ascorbate and other antioxidants, in combi-

nation with other protective factors found in hibernation

[10,51], in the treatment of heart disease and other

diseases of the periphery, as well as neurological disor-

ders, including cerebral ischemia and trauma.

Acknowledgments—The authors thank Shufen Wu for superb technicalassistance. This work was funded by NS-34115 and NS-40169 supportedby NINDS, NIMH, NCRR, NCMHD, and the NYU Ricciardi ResearchFund.

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ABBREVIATIONS

AGS—arctic ground squirrels

AO—L-ascorbate oxidase

Asc—ascorbate

[Asc]p—ascorbate concentration in plasma

DHA—dehydroascorbate

GSH—glutathione

GSSG—oxidized glutathione

MCA—monochloroacetic acid

[Urate]p—urate concentration in plasma